Sergio Mayor-Torroglosa

A computerized analysis of the entire retinal ganglion cell population and its spatial distribution in adult rats

In adult albino (SD) and pigmented (PVG) rats the entire population of retinal ganglion cells (RGCs) was quantified and their spatial distribution analyzed using a computerized technique. RGCs were back-labelled from the optic nerves (ON) or the superior colliculi (SCi) with Fluorogold (FG). Numbers of RGCs labelled from the ON [SD: 82,818+/-3,949, n=27; PVG: 89,241+/-3,576, n=6) were comparable to those labelled from the SCi [SD: 81,486+/-4,340, n=37; PVG: 87,229+/-3,199; n=59]. Detailed methodology to provide cell density information at small scales demonstrated the presence of a horizontal region in the dorsal retina with highest densities, resembling a visual streak.

Retinal ganglion cell death after acute retinal ischemia is an ongoing process whose severity and duration depends on the duration of the insult.

In adult Sprague-Dawley rats we have investigated retinal ganglion cell survival after transient intervals of retinal ischemia of 30, 45, 60, 90 or 120 min duration, induced by ligature of the ophthalmic vessels. Animals were killed 5, 7, 14, 21, 30, 60, 90 or 180 days later and densities of surviving retinal ganglion cells were estimated in retinal whole mounts by counting cells labelled with diAsp. This dye was applied, 3 days prior to death, to the ocular stump of the intraorbitally transected optic nerve. We found that retinal ganglion cell loss after retinal ischemia proceeds for different lengths of time. All the ischemic intervals induced loss of retinal ganglion cells whose severity and duration was related to the length of the ischemic interval. Following 30 or 45 min of ischemia, cell loss lasted 14 days and caused the death of 46 or 50%, respectively, of the population of retinal ganglion cells. Sixty, 90 or 120 min of retinal ischemia were followed by a period of cell loss that lasted up to 90 days and caused the death of 75%, 87% or 99%, respectively, of the population of retinal ganglion cells. We conclude that retinal ganglion cell loss after retinal ischemia is an ongoing process that may last up to 3 months after the injury and that its severity and duration are determined by the ischemic interval.

Ocular hypertension impairs optic nerve axonal transport leading to progressive retinal ganglion cell degeneration.

Ocular hypertension (OHT) is the main risk factor of glaucoma, a neuropathy leading to blindness. Here we have investigated the effects of laser photocoagulation (LP)-induced OHT, on the survival and retrograde axonal transport (RAT) of adult rat retinal ganglion cells (RGC) from 1 to 12 wks. Active RAT was examined with fluorogold (FG) applied to both superior colliculi (SCi) 1 wk before processing and passive axonal diffusion with dextran tetramethylrhodamine (DTMR) applied to the optic nerve (ON) 2 d prior to sacrifice. Surviving RGCs were identified with FG applied 1 wk pre-LP or by Brn3a immunodetection. The ON and retinal nerve fiber layer were examined by RT97-neurofibrillar staining. RGCs were counted automatically and color-coded density maps were generated. OHT retinas showed absence of FG+ or DTMR+RGCs in focal, pie-shaped and diffuse regions of the retina which, by two weeks, amounted to, approximately, an 80% of RGC loss without further increase. At this time, there was a discrepancy between the total number of surviving FG-prelabelled RGCs and of DMTR+RGCs, suggesting that a large proportion of RGCs had their RAT impaired. This was further confirmed identifying surviving RGCs by their Brn3a expression. From 3 weeks onwards, there was a close correspondence of DTMR+RGCs and FG+RGCs in the same retinal regions, suggesting axonal constriction at the ON head. Neurofibrillar staining revealed, in ONs, focal degeneration of axonal bundles and, in the retinal areas lacking backlabeled RGCs, aberrant staining of RT97 characteristic of axotomy. LP-induced OHT results in a crush-like injury to ON axons leading to the anterograde and protracted retrograde degeneration of the intraocular axons and RGCs.

Death and neuroprotection of retinal ganglion cells after different types of injury

In adult Sprague—Dawley rats, retinal ganglion cell survival was investigated after intraorbital optic nerve section and after transient ischemia of the retina induced by elevation of the intraocular pressure or by selective ligature of the ophthalmic vessels. The thickness of the inner nuclear and inner plexiform layers was also assessed after transient periods (120 min) of retinal ischemia induced by selective ligature of the ophthalmic vessels. In addition, we have also investigated the neuroprotective effects of different substances in these paradigms. The intraocular injection of brain-derived neurotrophic factor increased RGC survival after retinal ischemia induced by elevation of the intraocular pressure or by selective ligature of the ophthalmic vessels. The caspase inhibitor Z-DEVD increased retinal ganglion cell survival after optic nerve section and also after 90 min of retinal ischemia induced by selective ligature of the ophthalmic vessels. The peptide Bcl-2 did not increase retinal ganglion cell survival after optic nerve section but increased retinal ganglion cell survival after 60 or 90 min of retinal ischemia induced by selective ligature of the ophthalmic vessels. Finally, BDNF, nifedipine, naloxone and bcl-2 prevented in part the decrease in thickness of the inner nuclear layer and inner plexiform layer induced by selective ligature of the ophthalmic vessels. Our results suggest that retinal ganglion cell loss induced by different types of injury, may be prevented by substances with neuroprotective effects, by altering steps of the cascade of events leading to cell death.

Transient ischemia of the retina results in massive degeneration of the retinotectal projection: long-term neuroprotection with brimonidine.

In adult rats, we have induced retinal ischemia and investigated anterogradely labeled surviving retinal ganglion cell (RGC) afferents to the contralateral superior colliculus (SC). The animals received topically in their left eyes two 5-microl drops of saline or saline-containing 0.5% brimonidine (BMD), 1 h before 90 min of retinal ischemia induced by ligature of the left ophthalmic vessels. Two months after ischemia, the anterogradely transported neuronal tracer cholera toxin B subunit (CTB) was injected in the ischemic eyes and animals were processed 4 days later. As controls and for comparison, the retinotectal innervation of unlesioned age-matched control rats was also examined with CTB. In control and experimental animals, serial coronal sections of the mesencephalon and brainstem were immunoreacted for CTB and the area and thickness of the two most superficial layers of the SC containing densely CTB-labeled profiles were estimated with an image analysis system. Ninety minutes of ischemia resulted 2 months later in reduced density of CTB-labeled profiles in the contralateral SC of the vehicle-treated rats, representing less than one half the area occupied by CTB-labeled profiles in control rats. This resulted in shrinkage of these layers and in the presence of areas virtually devoid of CTB immunoreactivity, suggesting orthograde degeneration of retinal terminals and/or decrease of anterograde axonal transport. Topical pretreatment with BMD resulted 2 months later in CTB immunoreactivity that occupied the superficial layers of the contralateral SC in an area of approximately 86% of that observed in the unlesioned control group of animals, indicating that BMD protects against ischemia-induced degeneration of the retinotectal projection, and preserves anterograde axonal transport.

Neuroprotection of retinal ganglion cell function and their central nervous system targets

Technological advances have enabled the observation of a large number of retinal ganglion cells (RGCs) in an objective manner. In animal models, it has been shown how retinal ischaemia induces profound functional and structural alterations of the inner retinal and RGC layers by 3 months. These findings reflect degeneration of the inner retinal layers, the RGC population and of the retinotectal projection. Functionally, this implies a permanent disconnection of the retina from its main retinorecipient target region in the brain. Brimonidine, a selective α-2 adrenergic agonist, has been shown to activate α-2 adrenergic receptors in the retina and promote the survival and function of RGCs post-injury. This agent may prevent or diminish ischaemia-induced alterations in the inner and RGC areas as well as in the main retinofugal projection. Understanding the pattern of degeneration that occurs in the major retinofugal pathway following retinal ischaemia will benefit ongoing studies conducted to develop neuroprotectant-based treatment strategies for progressive neuropathies such as glaucoma.